Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins

✍ Scribed by Eiji Kinoshita; Atsushi Yamada; Hironori Takeda; Emiko Kinoshita-Kikuta; Tohru Koike

• Book ID: 102925572
• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 696 KB
• Volume: 28
• Category: Article
• ISSN: 1615-9306
• DOI: 10.1002/jssc.200401833

✦ Synopsis

Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the separation of natural or artificial products that is beginning to find industrial applications. Here, we introduce a novel procedure for the separation of phosphopeptides and phosphorylated proteins by immobilized zinc(II) affinity chromatography. The phosphate-binding site of the affinity gel is an alkoxide-bridged dinuclear zinc(II) complex, the 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex (Phos-tag), which is linked to a highly cross-linked 4% (w/v) agarose. The affinity gel (Phos-tag agarose) was prepared by the quantitative reaction of N-hydroxysuccinimide-activated Sepharose and a Phos-tag derivative having a 2-aminoethylcarbamoyl group in dry CH 3 CN. Phosphopeptides were retrieved in a quantitative and highly selective manner by a spin column method using Phos-tag agarose at room temperature. Furthermore, in this study, we demonstrate a simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, a s1 -casein, and b-casein at physiological pH.